Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

Abstract

A 35-70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contained a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton L chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin had identical staining patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also had similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity was markedly increased after phosphorylation. The actin-activiated ATPase activity of phosphorylated myosin was inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin resulted in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.